1. An Introduction to Cloning and
Recombinant DNA
Ankit Bhardwaj

2. What Are Clones?
 Clones
• Genetically identical molecules, cells, or
organisms all derived from a single ancestor
 Cloning
• The production of identical copies of molecules,
cells, or organisms from a single ancestor

3. Cloning Higher Plants and Animals
 Development of methods for cloning higher
plants and animals represents a significant
advance in genetic technology
• Improving crops
• Producing domestic animals

4. Plants Can Be Cloned
from Single Cells
 1950s: Charles Steward grew individual carrot
cells in the laboratory by using special nutrients
 Single cells grew and divided to form a ball of
undifferentiated cells (callus)
 Calluses transferred to a different medium grew
into full-size carrots (clones)

5. A Cloned Plant
 Single cells grow and divide to form a callus

6. Animals Can Be Cloned
by Several Methods
 Embryo splitting
• After in vitro fertilization, early embryonic cells are
divided and grown into clones
 Nuclear transfer (cell fusion)
• Enucleated eggs are fused with embryonic or
adult cells and grown into clones
• Dolly the sheep

7. Why is DNA Cloning Important?
 DNA clones are used to find genes, map them,
and transfer them between species
 Cloning technology is used to find carriers of
genetic disorders, perform gene therapy, and
create disease-resistant plants

8. Cloning Genes
Is a Multistep Process
 Technology was developed to clone segments of
DNA molecules, based on enzymes (restriction
endonucleases) that recognize and cut DNA at
specific nucleotide sequences

9. Recombinant DNA Technology
 Recombinant DNA technology
• Techniques in which DNA fragments are linked to
self-replicating vectors to create recombinant
DNA molecules which are replicated in a host cell

10. What’s Needed to Clone DNA?
 A way to cut DNA at specific sites
 A carrier molecule to hold DNA for cloning
 A place where the DNA can be copied (cloned)

11. DNA Can Be Cut at Specific Sites
Using Restriction Enzymes
 Bacteria produce restriction enzymes to protect
themselves from viral infections
 Restriction enzymes
• Bacterial enzymes that cut DNA at specific sites

12. Vectors are Carriers of DNA to be Cloned
 Linking DNA segments produced by restriction-
enzymes with vectors (plasmids or engineered
viral chromosomes) produces recombinant DNA
 Vectors
• Self-replicating DNA molecules used to transfer
foreign DNA segments between host cells

13. Cloning Recombinant DNA
 Recombinant DNA molecules are transferred
into host cells; cloned copies are produced as
the host cells grow and divide
 Most common host cell: the bacterium E. coli
 Cloned DNA molecules can be recovered from
the host cells and purified for further use

14. E. coli
 The recognition and cutting site for EcoRI

15. Plasmids
 Plasmids used as vectors for cloning DNA

16. Steps in the Process of Cloning
 DNA is cut with a restriction enzyme
• Fragments produced end in specific sequences
 Fragments are mixed with vector molecules cut
by the same enzyme
• DNA ligase joins recombinant DNA molecules

17. Steps in the Process of Cloning
 Plasmid vectors with inserted DNA fragments
are transferred into bacterial cells
• Recombinant plasmids replicate and produce
many clones of the recombinant DNA molecule
• Colonies carrying cloned recombinant DNA
molecules are identified, collected, and grown
• Host cells are broken open and recombinant
plasmids are extracted

18. Cloning

19. Cloning Bacteria on Petri Plates
 Each colony is a clone, descended from a single
cell

20. Introduction in host cell

21. Identifying Colonies
With Recombinant DNA
 Plasmid pBR322 has been engineered to carry
two antibiotic-resistance genes with restriction
sites, one for tetracycline, one for ampicillin
 Colonies with human DNA inserted into the
tetracycline gene will not grow on tetracycline
plates, but will grow on ampicillin plates

23. 13.5 A Revolution in Cloning:
The Polymerase Chain Reaction
 Polymerase chain reaction (PCR)
• A method for amplifying DNA segments using
cycles of denaturation, annealing to primers, and
DNA polymerase-directed DNA synthesis
 PCR copies a DNA molecule without restriction
enzymes, vectors, or host cells
• Faster and easier than conventional cloning

24. First Step in PCR: Denaturation
1. DNA is heated to break the hydrogen bonds
between the two polynucleotide strands
• Two single-stranded DNA molecules serve as
templates

25. Second Step in PCR: Annealing
2. Short nucleotide sequences (primers for DNA
replication) are mixed with the DNA and bind to
complementary regions on single-stranded DNA
• Takes place at lower temperature
• Primers are 20-30 nucleotides long, synthesized
in the laboratory

26. Third Step in PCR: DNA Synthesis
3. The enzyme Taq polymerase is added to
synthesize a complementary DNA strand
• Taq is a DNA polymerase from a bacterium found
in hot springs
 These three steps make up one PCR cycle

27. Many Uses for PCR
 DNA to be amplified by PCR does not have to
be purified and can be present in small amounts
• Used in clinical diagnosis, forensics, conservation
• Samples can be small or old (insects in amber)

28. THANK YOU
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